Starch debranching enzymes

ABSTRACT

The invention relates to a genetically engineered variant of a parent starch debranching enzyme, i.e. a pullulanase or an isoamylase, the enzyme variant having an improved thermostability at a pH in the range of 4-6 compared to the parent enzyme and/or an increased activity towards amylopectin and/or glycogen compared to the parent enzyme, to methods for producing such starch debranching enzyme variants with improved thermostability and/or altered substrate specificity, and to a method for converting starch to one or more sugars using at least one such enzyme variant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/375,720, filed on Feb. 26, 2003 (now U.S. Pat. No. 7,374,922), whichis a continuation of U.S. application Ser. No. 09/833,435, filed on Apr.12, 2001 (abandoned), which is a continuation of U.S. application Ser.No. 09/346,237, filed Jul. 1, 1999 (now U.S. Pat. No. 6,265,197), whichclaims priority under 35 U.S.C. 119 of Danish application PA 1998 00868,filed Jul. 2, 1998, and the benefit of U.S. provisional application No.60/094,353, filed on Jul. 28, 1998, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel starch debranching enzymes, inparticular pullulanases and isoamylases, designed for use in a starchconversion process comprising a liquefaction step and a saccharificationstep, as well as to the production of such enzymes and the use of suchenzymes in a starch conversion process.

BACKGROUND OF THE INVENTION

Starches such as corn, potato, wheat, manioc and rice starch are used asthe starting material in commercial large scale production of sugars,such as high fructose syrup, high maltose syrup, maltodextrins, amylose,G4-G6 oligosaccharides and other carbohydrate products such as fatreplacers.

Degradation of Starch

Starch usually consists of about 80% amylopectin and 20% amylose.Amylopectin is a branched polysaccharide in which linear chains α-1,4D-glucose residues are joined by α-1,6 glucosidic linkages. Amylopectinis partially degraded by α-amylase, which hydrolyzes the1,4-α-glucosidic linkages to produce branched and linearoligosaccharides. Prolonged degradation of amylopectin by α-amylaseresults in the formation of so-called α-limit dextrins which are notsusceptible to further hydrolysis by the α-amylase. Branchedoligosaccharides can be hydrolyzed into linear oligosaccharides by adebranching enzyme. The remaining branched oligosaccharides can bedepolymerized to D-glucose by glucoamylase, which hydrolyzes linearoligosaccharides into D-glucose.

Amylose is a linear polysaccharide built up of D-glucopyranose unitslinked together by α-1,4 glucosidic linkages. Amylose is degraded intoshorter linear oligosaccharides by α-amylase, the linearoligosaccharides being depolymerized into D-glucose by glucoamylase.

In the case of converting starch into a sugar, the starch isdepolymerized. The depolymerization process consists of a pretreatmentstep and two or three consecutive process steps, namely a liquefactionprocess, a saccharification process and, depending on the desired endproduct, optionally an isomerization process.

Pre-Treatment of Native Starch

Native starch consists of microscopic granules which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be handled. This reduction in viscosity is today mostlyobtained by enzymatic degradation.

Liquefaction

During the liquefaction step, the long-chained starch is degraded intosmaller branched and linear units (maltodextrins) by an α-amylase (e.g.Termamyl™, available from Novo Nordisk A/S, Denmark). The liquefactionprocess is typically carried out at about 105-110° C. for about 5 to 10minutes followed by about 1-2 hours at about 95° C. The pH generallylies between about 5.5 and 6.2. In order to ensure an optimal enzymestability under these conditions, calcium is added, e.g. 1 mM of calcium(40 ppm free calcium ions). After this treatment the liquefied starchwill have a “dextrose equivalent” (DE) of 10-15.

Saccharification

After the liquefaction process the maltodextrins are converted intodextrose by addition of a glucoamylase (e.g. AMG™, available from NovoNordisk A/S) and a debranching enzyme, such as an isoamylase (see e.g.U.S. Pat. No. 4,335,208) or a pullulanase (e.g. Promozyme™, availablefrom Novo Nordisk A/S) (see U.S. Pat. No. 4,560,651). Before this stepthe pH is reduced to a value below 4.5, e.g. about 3.8, maintaining thehigh temperature (above 95° C.) for a period of e.g. about 30 min. toinactivate the liquefying α-amylase to reduce the formation of shortoligosaccharides called “panose precursors” which cannot be hydrolyzedproperly by the debranching enzyme.

The temperature is then lowered to 60° C., glucoamylase and debranchingenzyme are added, and the saccharification process proceeds for about24-72 hours.

Normally, when denaturing the α-amylase after the liquefaction step, asmall amount of the product comprises panose precursors which cannot bedegraded by pullulanases or AMG. If active amylase from the liquefactionstep is present during saccharification (i.e. no denaturing), this levelcan be as high as 1-2% or even higher, which is highly undesirable as itlowers the saccharification yield significantly. For this reason, it isalso preferred that the α-amylase is one which is capable of degradingthe starch molecules into long, branched oligosaccharides (such as,e.g., the Fungamyl™-like α-amylases) rather than shorter branchedoligosaccharides.

Isomerization

When the desired final sugar product is e.g. high fructose syrup, thedextrose syrup may be converted into fructose by enzymaticisomerization. After the saccharification process the pH is increased toa value in the range of 6-8, preferably about pH 7.5, and the calcium isremoved by ion exchange. The dextrose syrup is then converted into highfructose syrup using, e.g., an immobilized glucose isomerase (such asSweetzyme™, available from Novo Nordisk A/S).

Debranching Enzymes

Debranching enzymes which can attack amylopectin are divided into twoclasses: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41),respectively. Isoamylase hydrolyses α-1,6-D-glucosidic branch linkagesin amylopectin and β-limit dextrins and can be distinguished frompullulanases by the inability of isoamylase to attack pullulan, and bytheir limited action on α-limit dextrins.

When an acidic stabilised “Termamyl™-like” α-amylase is used for thepurpose of maintaining the amylase activity during the entiresaccharification process (no inactivation), the degradation specificityshould be taken into consideration. It is desirable in this regard tomaintain the α-amylase activity throughout the saccharification process,since this allows a reduction in the amyloglucidase addition, which iseconomically beneficial and reduces the AMG™ condensation productisomaltose, thereby increasing the DE (dextrose equivalent) yield.

It will be apparent from the above discussion that the known starchconversion processes are performed in a series of steps, due to thedifferent requirements of the various enzymes in terms of e.g.temperature and pH. It would therefore be desirable to be able toengineer one or more of these enzymes so that the overall process couldbe performed in a more economical and efficient manner. One possibilityin this regard is to engineer the otherwise thermolabile debranchingenzymes so as to render them more stable at higher temperatures. Thepresent invention relates to such thermostable debranching enzymes, theuse of which provides a number of important advantages which will bediscussed in detail below. It also relates to starch debranching enzymeswith an altered substrate specificity.

SUMMARY OF THE INVENTION

An object of the present invention is thus to provide thermostabledebranching enzymes, for example pullulanases and isoamylases, which aresuitable for use at high temperatures in a starch conversion process, inparticular using genetic engineering techniques in order to identify andsynthesize suitable enzyme variants. Another object of the invention isto provide novel starch debranching enzymes with an altered substratespecificity.

In its broadest aspect, the present invention can thus be characterizedas relating to novel starch debranching enzymes with improved propertiesin terms of e.g. thermostability or substrate specificity, as well asmethods for producing such enzymes and the use of such enzymes in astarch conversion process.

In one particular aspect, the invention relates to a geneticallyengineered variant of a parent starch debranching enzyme, the enzymevariant having an improved thermostability at a pH in the range of 4-6compared to the parent enzyme.

Another aspect of the invention relates to a genetically engineeredvariant of a parent starch debranching enzyme, the enzyme variant havingan increased activity towards amylopectin and/or glycogen compared tothe parent enzyme.

A further aspect of the invention relates to a method for producing astarch debranching enzyme variant with increased thermostability, themethod comprising the steps of:

-   -   identifying one or more amino acid residues and/or amino acid        regions associated with thermostability in a first parent starch        debranching enzyme,    -   identifying one or more homologous amino acid residues and/or        amino acid regions in a second parent starch debranching enzyme        by means of alignment of the amino acid sequences of the first        and second parent starch debranching enzymes, and    -   mutating one or more of the homologous amino acid residues        and/or amino acid regions in the second parent starch        debranching enzyme to produce an enzyme variant with increased        thermostability.

A still further aspect of the invention relates to a method forproducing a starch debranching enzyme variant with altered substratespecificity, the method comprising the steps of:

-   -   identifying one or more amino acid residues in at least one        amino acid loop associated with specificity towards a desired        substrate in a first parent starch debranching enzyme,    -   identifying one or more homologous amino acid residues in at        least one corresponding loop in a second parent starch        debranching enzyme by means of alignment of the amino acid        sequences of the first and second parent starch debranching        enzymes, and    -   mutating one or more of the homologous amino acid residues in at        least one loop in the second parent starch debranching enzyme to        produce an enzyme variant with altered substrate specificity.

The term “loop” means, at least in the context of the present invention,the sequence part following the beta-strand/sheet part of the sequencein question. Said “beta strands/sheets” may be identified by multiplesequence alignment of sequences of the present invention and sequenceswith a known three dimensional structure. Such alignments can be madeusing standard alignment programs, available from e.g. the UWGCG package(Program Manual for the Wisconsin Package, Version 8, August 1994,Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711).

Known three-dimensional enzyme structures are available from BrookhavenDatabank. Examples of such are the three-dimensional structure of theAspergillus oryzae TAKA α-amylase (Swift et al., 1991), the Aspergillusniger acid amylase (Brady et al, 1991), the structure of pig pancreaticα-amylase (Qian et al., 1993), and the barley α-amylase (Kadziola et al.1994, Journal of Molecular Biology 239:104-121; A. Kadziola, Thesis,Dept of Chemistry, U. of Copenhagen, Denmark).

The invention relates in addition to a method for converting starch toone or more sugars, the method comprising debranching the starch usingat least one enzyme variant as described herein.

DETAILED DESCRIPTION OF THE INVENTION

In the present context, the term “thermostable” refers in general to thefact that the debranching enzyme variants according to the inventionhave an improved thermostability compared to the relevant parent enzyme.The degree of improvement in thermostability can vary according tofactors such as the thermostability of the parent enzyme and theintended use of the enzyme variant, i.e. whether it is primarilyintended to be used in for liquefaction or for saccharification or both.It will be apparent from the discussion below that for saccharification,the enzyme variant should maintain a substantial degree of enzymeactivity during the saccharification step at a temperature of at leastabout 63° C., preferably at least about 70° C., while an enzyme variantdesigned for use in the liquefaction step should be able to maintain asubstantial degree of enzyme activity at a temperature of at least about95° C.

The improved thermostability of enzyme variants according to theinvention can in particular be defined according to one or more of thefollowing criteria:

In one embodiment, the enzyme variant of the invention has an improvedthermostability as defined by differential scanning calorimetry (DSC)using the method described below.

In another embodiment, the enzyme variant of the invention has animproved thermostability as defined by an increased half-time (T_(1/2))of at least about 5%, preferably at least about 10%, more preferably atleast about 15%, more preferably at least about 25%, most preferably atleast about 50%, such as at least 100%, in the “T_(1/2), assay forliquefaction” described herein, using a pH of 5.0 and a temperature of95° C. Enzyme variants according to this definition are suitable for usein the liquefaction step of the starch conversion process.

Alternatively or additionally, an enzyme variant suitable for use inliquefaction can be defined as having an improved thermostability asdefined by an increased residual enzyme activity of at least about 5%,preferably at least about 10%, more preferably at least about 15%, morepreferably at least about 25%, most preferably at least about 50%, inthe “assay for residual activity after liquefaction” described herein,using a pH of 5.0 and a temperature of 95° C.

In a further embodiment, the enzyme variant of the invention has animproved thermostability as defined by an increased half-time (T_(1/2))of at least about 5%, preferably at least about 10%, more preferably atleast about 15%, more preferably at least about 25%, most preferably atleast about 50%, such as at least 100%, in the “T_(1/2) assay forsaccharification” described herein, using a pH of 4.5 and a temperatureof 70° C. Such variants are suitable for use in the saccharificationstep of the starch conversion process.

Alternatively or additionally, an enzyme variant suitable forsaccharification can be defined as having an improved thermostability asdefined by an increased residual enzyme activity of at least about 5%,preferably at least about 10%, more preferably at least about 15%, morepreferably at least about 25%, most preferably at least about 50%, inthe “assay for residual activity after saccharification” describedherein, using a pH of 4.5 and a temperature of 63° C. Preferably, thisimproved thermostability is also observed when assayed at a temperatureof 70° C.

The term “substantially active” as used herein for a given enzymevariant and a given set of conditions of temperature, pH and time meansthat the relative enzymatic activity of the enzyme variant is at leastabout 25%, preferably at least about 50%, in particular at least about60%, especially at least about 70%, such as at least about 90% or 95%,e.g. at least about 99% compared to the relative activity of the parentenzyme under the given set of conditions mentioned in connection withimproved thermostability right above.

An enzyme variant “derived from” a given enzyme (a “parent enzyme”)means that the amino acid sequence of the parent enzyme has beenmodified, i.e. by substitution, deletion, insertion and/or loop transferas described below, to result in the enzyme variant. In the case of aparent enzyme produced by an organism such as a microorganism, where anenzyme variant according to the invention is derived from the parentenzyme, the enzyme variant may be produced by appropriate transformationof the same or a similar microorganism or other organism used to producethe parent enzyme.

One advantage of the thermostable debranching enzymes of the inventionis that they make it possible to perform liquefaction and debranchingsimultaneously before the saccharification step. This has not previouslybeen possible, since the known pullulanases and isoamylases withacceptable specific activity are thermolabile and are inactivated attemperatures above 60° C. (Some thermostable pullulanases fromPyrococcus are known, but these have an extremely low specific activityat higher temperatures and are thus unsuitable for purposes of thepresent invention). By debranching, using the thermostable debranchingenzymes of the invention, during liquefaction together with the actionof an α-amylase, the formation of panose precursors is reduced, therebyreducing the panose content in the final product and increasing theoverall saccharification yield. It is also possible in this manner toextend the liquefaction process time without risking formation of largeamount of panose precursors. By prolonging the liquefaction step, the DEyield is increased from 10-15 to e.g. 15-20, reducing the need forglucoamylase. This reduced glucoamylase requirement is in turnadvantageous as the formation of undesired isomaltose is reduced,thereby resulting in an increased glucose yield. In addition, thereduced glucoamylase addition enables the saccharification step to becarried out at a higher substrate concentration (higher DS, drysubstances, concentration) than the normal approx. 30-35% used accordingto the prior art. This allows reduced evaporation costs downstream, e.g.in a high fructose corn syrup process, and the saccharification reactiontime can also be reduced, thereby increasing production capacity. Afurther advantage is that α-amylase used in the liquefaction processdoes not need to be inactivated/denatured in this case.

Furthermore, it is also possible to use the thermostable debranchingenzymes according to the invention during saccharification, which isadvantageous for several reasons. In the conventional starchsaccharification process, the process temperature is not more than 60°C. due to the fact that neither the saccharification enzyme pullulanasenor AMG™ are sufficiently thermostable to allow the use of a highertemperature. This is a disadvantage, however, as it would be verydesirable to run the process at a temperature of above about 60° C., inparticular above 63° C., e.g. about 70° C., to reduce microbial growthduring the relatively long saccharification step. Furthermore, a higherprocess temperature normally gives a higher activity per mg of enzyme(higher specific activity), thereby making it possible to reduce theweight amount of enzyme used and/or obtain a higher total enzymaticactivity. A higher temperature can also result in a higher dry mattercontent after saccharification, which would be beneficial in terms ofreducing evaporation costs.

Although a thermostable isoamylase might be regarded as being morebeneficial than a thermostable pullulanase when used in the liquefactionprocess, since isoamylases are characterised by their specificitytowards amylopectin and activity on higher molecular weight dextrins, apreferred alternative is to alter the specificity of a pullulanase so asto be more “isoamylase-like” in the sense of having improved activitytowards longer, branched-chain dextrins. Among the various pullulanasesthere are substantial differences in this respect, even among thepullanases of the same Bacillus origin.

Methods for Determining Stability and Activity

Thermostability

Thermostability of pullulanases and isoamylases can be detected bymeasuring the residual activity by incubating the enzyme underaccelerated stress conditions, which comprise: pH 4.5 in a 50 mM sodiumacetate buffer without a stabilizing dextrin matrix (such as theapproximately 35% dry matter which is normally present duringsaccharification). The stability can be determined at isotherms of e.g.63° C., 70° C., 80° C., 90° C. and 95° C., measuring the residualactivity of samples taken from a water bath at regular intervals (e.g.every 5 or 10 min.) during a time period of 1 hour. For determiningstability for the purpose of liquefaction, a pH of 5.0, a temperature of95° C. and a total assay time of 30 minutes are used (“assay forresidual activity after liquefaction”). For determining stability forthe purpose of saccharification, a pH of 4.5, a temperature of 63° C. or70° C. and a total assay time of 30 minutes are used (“assay forresidual activity after saccharification”).

Alternatively, the thermostability may be expressed as a “half-time”(T_(1/2)), which is defined as the time, under a given set ofconditions, at which the activity of the enzyme being assayed is reducedto 50% of the initial activity at the beginning of the assay. In thiscase, the “T_(1/2), assay for liquefaction” uses a pH of 5.0 and atemperature of 95° C., while the “T_(1/2) assay for saccharification”uses a pH of 4.5 and a temperature of 70° C. The assay is otherwiseperformed as described above for the respective assays for residualactivity.

Activity: Somogyi-Nelson Method for Determination of Reducing Sugars

The activity of both pullulanases and isoamylases can be measured usingthe Somogyi-Nelson method for the determination of reducing sugars (J.Biol. Chem. 153, 375 (1944)). This method is based on the principle thatsugar reduces cupric ions to cuprous oxide, which reacts with anarsenate molybdate reagent to produce a blue colour that is measuredspectrophotometrically. The solution to be measured must contain 50-600mg of glucose per liter. The procedure for the Somogyi-Nelson method isas follows:

Sample value: Pipette 1 ml of sugar solution into a test tube. Add 1 mlof copper reagent. Stopper the test tube with a glass bead. Place thetest tube in a boiling water bath for 20 minutes. Cool the test tube.Add 1 ml of Nelson's colour reagent. Shake the test tube withoutinverting it. Add 10 ml of deionized water. Invert the test tube andshake vigorously. Measure the absorbance at 520 nm, inverting the testtube once immediately prior to transfer of the liquid to the cuvette.

Blank value: Same procedure as for the sample value, but with waterinstead of sugar solution.

Standard value: Same procedure as for the sample value.

Calculations

In the region 0-2 the absorbance is proportional to the amount of sugar.

$\begin{matrix}{{{mg}\mspace{14mu}{sugar}\text{/}1} = \frac{100\mspace{14mu}\left( {{sample} - {blank}} \right)}{\left( {{standard} - {blank}} \right)}} \\{{\%\mspace{14mu}{glucose}} = \frac{\left( {{sample} - {blank}} \right)}{100 \times \left( {{standard} - {blank}} \right)}}\end{matrix}$Reagents1. Somogyi's Copper Reagent

35.1 g Na₂HPO₄.2H₂O and 40.0 g potassium sodium tartrate (KNaC₄H₄.4H₂O)are dissolved in 700 ml of deionized water. 100 ml of 1N sodiumhydroxide and 80 ml of 10% cupric sulphate (CuSO₄.5H₂O) are added. 180 gof anhydrous sodium sulphate are dissolved in the mixture, and thevolume is brought to 1 liter with deionized water.

2. Nelson's Colour Reagent

50 g of ammonium molybdate are dissolved in 900 ml of deionized water.Then 42 ml of concentrated sulphuric acid are added, followed by 6 g ofdisodium hydrogen arsenate heptahydrate dissolved in 50 ml of deionizedwater, and the volume is brought to 1 liter with deionized water. Thesolution is allowed to stand for 24-48 hours at 37° C. before use and isstored in the dark in a brown glass bottle with a glass stopper.

3. Standard

100 mg of glucose (anhydrous) are dissolved in 1 liter of deionizedwater.

Substrate Specificity

Methods for the determination and characterisation of the profile ofaction and specificity of pullulanases and isoamylases for varioussubstrates (e.g. amylopectin, glycogen and pullulan) are described byKainuma et al. in Carbohydrate Research, 61 (1978) 345-357. Using thesemethods, the relative activity of an isoamylase or a pullulanase can bedetermined, and the relative activity of an enzyme variant according tothe invention compared to the relative activity of the parent enzyme canbe assessed, for example to determine whether a pullulanase variant hasthe desired increased specificity toward high molecular weightsaccharides such as amylopectin compared to the parent enzyme.

Starch Conversion

As indicated above, in one embodiment of the invention, the starchconversion process comprises debranching using a thermostabledebranching enzyme of the invention during the liquefaction steptogether with an α-amylase. The liquefaction step is typically carriedout at a pH between 4.5 and 6.5, e.g. from 5.0 to 6.2, at a temperaturein the range of 95-110° C. for a period of 1 to 3 hours, preferablyabout 1.5-2 hours. It is preferred, however, that the pH is as low aspossible, e.g. from 4.5 to 5.0, as long as the enzyme(s) used for theliquefaction have a sufficient stability at the pH in question. If theα-amylase is calcium dependent, calcium may be added in an amount offrom 30 to 50 ppm, such as around 40 ppm (or 0.75 to 1.25 mM, such asaround 1 mm) in the liquefaction step to stabilise the enzyme. Asexplained above, the α-amylase need not be inactivated after theliquefaction step to reduced the panose formation in this case.

Examples of specific α-amylases which can be used in the liquefactionstep include Bacillus licheniformis α-amylases, such as the commerciallyavailable products Termamyl®, Spezyme® AA, Spezyme® Delta AA, Maxamyl®and Kleistase®, and the α-amylase mutants described in WO 96/23874 (NovoNordisk) and PCT/DK97/00197 (Novo Nordisk).

Isoamylases which can be used as a parent enzyme according to theinvention include, but are not limited to, the thermostable isoamylasederived from the thermophilic acrhaebacterium Sulfolobus acidocaldarius(Maruta, K et al., (1996), Biochimica et Biophysica Acta 1291, p.177-181), isoamylase from Rhodothermus marinus (e.g. the isoamylase ofSEQ ID NO 3) and isoamylase from Pseudomonas, e.g. Pseudomonasamyloderamosa (e.g. Pseudomonas amyloderamosa isoamylase disclosed inEMBL database accession number J03871 or GeneBank accession numberN90389).

Examples of pullulanases which can be used as a parent enzyme include,but are not limited to, a thermostable pullulanase from e.g. Pyrococcusor a protein engineered pullulanase from e.g. a Bacillus strain such asBacillus acidopullulyticus (e.g. Promozyme™ or SEQ ID NO 1) or Bacillusderamificans (e.g. SEQ ID NO 2; or the Bacillus deramificans pullulanasewith GeneBank accession number Q68699).

While prior art methods for saccharification employ a temperature of notmore than about 60° C., the present invention provides thermostabledebranching enzymes that can remain active at higher temperatures, i.e.at least about 63° C. and preferably at least about 70° C. so as toeliminate possibilities for microbial growth. Examples of suitableglucoamylases for saccharification include Aspergillus nigerglucoamylases, such as ADG™. The saccharification process typicallyproceeds for about 24-72 hours at a pH of about 4.0-4.5, preferablyabout 4.0.

When the desired final sugar product is e.g. a high fructose syrup ofapprox. 50% fructose syrup, the formed D-glucose is isomerized by anisomerase at a pH around 6-8, preferably about 7.5. An example of asuitable isomerase is an glucose isomerase such as the glucose isomerasederived from Streptomyces murinus. The isomerase may be an immobilizedglucose isomerase, such as Sweetzyme®.

Calcium is normally removed if added before the liquefaction step.

Engineering of Pullulanases and Isoamylases

The pullulanases and isoamylases are members of the family 13 amylases(Henrissat, B. et al., Biochem J. 293:781-788, 1993). This suggests thatthey have the same overall structure in the central part of the moleculeconsisting of an A, B and C domain. The B domains vary quitedramatically in size and structure, whereas the other two domains arebelieved to generally possess a high degree of homology. The A domain iscomposed of an alpha-8/beta-8 structure (a beta-barrel) and 8 loopsbetween the beta-strands and the alpha-helices (a helix can in certaincases be absent, however). The sequences coming from the beta-strandpart of the beta-barrel point towards the substrate binding region.These regions are of particular interest for the specificity of theenzyme (Svensson, B. et al., Biochemical Society Transactions, Vol. 20;McGregor, J. Prot. Chem. 7:399, 1988). However, by using informationabout specific sequences and as well as general strategies for analyzingpullulanase and isoamylase sequences, the present invention provides thenecessary tools to be able to engineer these enzymes to produce variantswith improved thermostability and/or specificity.

Sequence Listings

The following sequence listings are referred to herein:

SEQ ID NO 1: pullulanase from Bacillus acidopullulyticus

SEQ ID NO 2: pullulanase from Bacillus deramificans

SEQ ID NO 3+ SEQ ID NO 12: isoamylase from Rhodothermus marinus

SEQ ID NO 4: isoamylase from Pseudomonas amyloderamosa JD270 (Chen, J Het al. (1990) Biochemica et Biophysica Acta 1087, pp 307-315)(Brookhaven database: 1BF2)

SEQ ID NO 5: pullulanase from Klebsiella pneumoniae (Kornacker et al.,Mol. Microbiol. 4:73-85 (1990))

SEQ ID NO 6: pullulanase from Klebsiella aerogenes (Katsuragi et al., J.Bacteriol. 169:2301-2306 (1987))

SEQ ID NO 7: isoamylase from Pseudomonas sp. SMP1 (Tognoni, A. et al.,U.S. Pat. No. 5,457,037)

SEQ ID NO 8: isoamylase from Favobacterium odoratum (JP 9623981, SusumuHizukuri et al.)

SEQ ID NO 9: isoamylase from Sulfolobus acidocaldarius, ATCC 33909(Biochimica et Biophysica Acta 1291 (1996) 177-181, Kazuhiko Maruta etal.)

SEQ ID NO 10: isoamylase from Sulfolobus solfataricus (GeneBankAccession no. Y08256).

SEQ ID NO 11: isoamylase from maize, Zea mays (ACCESSION U18908)

SEQ ID NO 13: Bacillus acidopullulyticus pulB gene (SEQ ID NO: 13).

Structure of Pullulanases

The appended FIG. 1 shows the amino acid sequence of four differentpullulanases as well as an alignment of these sequences. On the basis ofinformation provided by this alignment, it is possible to performhomology substitutions in order to obtain desired characteristics ofimproved thermostability and/or altered substrate specificity.

The four sequences are SEQ ID NO 5, 6, 1 and 2.

Structure of Isoamylases

The appended FIG. 2 shows the amino acid sequence of seven differentisoamylases as well as an alignment of these sequences. On the basis ofinformation provided by this alignment, it is possible to performhomology substitutions in order to obtain desired characteristics ofimproved thermostability and/or altered substrate specificity.

The seven sequences are SEQ ID NO 4, 7, 8, 9, 10, 3 and 11.

The X-ray structure of the Pseudomonas amyloderamosa isoamylase hasrecently been published in the Brookhaven database under number 1BF2.The structure confirms the overall view of the sequence alignmentmethod, but also shows certain differences to the suggested alignment.The corrected loop numbers deduced from the 3D structure of Pseudomonasamyloderamosa isoamylase (1BF2) are shown below:

Loop Suggested Deduced from structure 1. 210-230 211-231 2. 250-292251-295 3. 319-376 319-330 4. 401-436 401-436 5. 461-479 461-468 6.482-485 482-503 7. 530-539 533-580 8. 605-636 606-636Alignment of Pullulanases and Isoamylases

The appended FIG. 3 shows a “key alignment” of selected pullulanases andisoamylases (those of SEQ ID NO 1, 2, 3 and 4), in other words abest-fit alignment of homologous amino acid residues in the respectivesequences. The dashes (“----”) indicate presumed beta-strand positions.Each set of dashes is followed by a loop number, indicating the positionin the sequence of the loops. Information on the location of theindividual loops is found in Table 1 below.

By comparing the most homologous sequence from the “key alignment” oftwo or more relevant starch debranching enzymes with a new starchdebranching enzyme sequence, and aligning these two sequences, residuesfrom the new sequence homologous to residues in the sequence from thekey alignment can be determined. The homology may be found e.g. by usingthe GAP program from the UWGCG package (Program Manual for the WisconsinPackage, Version 8, August 1994, Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711). The new sequence can then be placed inthe key alignment by using a text editing program or other suitablecomputer program.

Table 1 below provides information on the location of selected regionsof interest in the various loops of the selected pullulanases andisoamylases, these loop regions being of general interest with regard tomodification to produce enzyme variants according to the invention. Loop3 below constitutes domain B (MacGregor, 1988), while the other loopsbelong to domain A.

TABLE 1 Loop 1 Loop 2 Loop 3 Loop 4 Loop 5 Loop 6 Loop 7 Loop 8Pullulanases Seq. Id. a. 369-397 419-458 484-525 553-568 582-608 613-616661-670 708-739 No. 1 b. 372-385 422-448 488-520 558-568 587-606 663-670710-724 c. 375-395 422-438 488-499 591-606 710-715 Seq. Id. a. 465-493515-554 580-621 649-664 680-711 714-717 757-765 804-834 No. 2Isoamylases Seq. Id. a. 210-230 250-292 319-376 401-437 461-479 482-485530-539 605-636 No. 4 Seq. Id. a. 176-195 218-257 283-334 359-381395-413 416-419 461-470 537-568 No. 3 b. 179-193 221-250 288-326 364-381399-411 463-470 539-553 c. 182-193 221-239 288-303 403-411 539-545

Where more than one region is listed for a given enzyme and loop inTable 1, the region listed first (i.e. a.) is in each case the expectedlength of the loop in question. The next region (i.e. b.) is thepreferred region for modification, and the last region (i.e. c.) is mostpreferred.

By performing modifications, i.e. substitutions, deletions, insertionsand/or loop transfer, in one or more these loops, engineered proteinshaving the desired properties in terms of improved thermostabilityand/or altered substrate specificity may be produced. An enzyme variantaccording to the invention may comprise any appropriate combination ofone or more substitutions, deletions, insertions and/or loop transfersto obtain the desired characteristics of improved thermostability and/oraltered activity.

The loop region of SEQ ID NO: 1, i.e. 369-397 (region denoted a), mayaccording to the invention suitably be replaced with the correspondingspatially placed region of SEQ ID NO:4, i.e. 176-195 (i.e. denoted a.).Further, region 371-385 of loop 1 (SEQ ID NO: 1) (i.e. denoted b.) maycorrespondingly be replaced with region 179-193 of loop 1 (SEQ ID NO. 4)(i.e. denoted b.).

In the present context, a simplified nomenclature is used to indicateamino acid substitutions in a given position. For example “G81P” refersto the substitution of a glycine residue in position 81 with a prolineresidue, and “F489G,A” refers to the substitution of a phenylalanineresidue in position 489 with either glycine or alanine.

Engineering for Improved Thermostability

When engineering for improved thermostability, either or both of thefirst and second parent enzymes may be an isoamylase or a pullulanase.For obtaining improved thermostability of isoamylases and pullulanases,we can focus especially on the B domain, which has been shown to beimportant for stability, using sequence homology information as furtherdescribed below.

Thermostability—Sequence Homology

Several different approaches may be used for the purpose of obtainingincreased thermostability, including proline substitutions, Gly to Alasubstitutions and Asn and Gln substitutions. Further details andexamples of these approaches are provided below.

Proline Substitutions

Proline substitutions, i.e. replacing one or more non-proline amino acidresidues with a proline residue, are suggested as an approach forobtaining thermostability on the basis of sequence alignment ofisoamylases and pullulanases. Examples of possible proline substitutionsare provided in the following.

Isoamylases

In P. amyloderamosa isoamylase (SEQ ID NO 4):

Positions for proline substitution include G81P, G99P, T18P, T199P,Q202P, T221, Q226P, A238P, T278P, R286P, A294P, G467P, G64P, V67P, E69P,A549P, G713P, T719P and D736P, and preferably S356P, T376P, T278P, N348Pand S454P.

In R. marinus Isoamylase (SEQ ID NO 3)

Positions for proline substitution include G154P, N305P and N669P, andpreferably R588P and K480P.

Pullulanases

In B. acidopullulyticus pullulanase (SEQ ID NO 1):

Preferred positions include A210P, V215P, L249P, K383P, S509P, T811P andG823P.

In B. deramificans Pullulanase (SEQ ID NO 2)

Preferred positions include G306P, V311P, L345P, D605P, T907P and A919P.

Gly to Ala Substitutions: for Example:

In P. amyloderamosa Isoamylase (SEQ ID NO 4)

G181A

Asn (N) and Gln (O) Substitutions

The new residues are chosen from all 20 possible amino acid residues,but preferably residues in a homologous position as seen from sequencealignment, Leu, Ile, Phe, Ala, Thr, Ser and Tyr being preferred. Ofspecial interest are the following:

SEQ ID NO 1:

Loop1: N379, N384

Loop2: N426, Q432, N434, N437, N444, N446

Loop3: N486, N490, Q502, N512, N515, N521

Loop4: -

Loop5: Q596

Loop6: N616, N621, Q628

Loop7: N679, N681, Q684

Loop8: N720, N722, N731, Q732

SEQ ID NO 2:

Loop1: N475, N480

Loop2: N522, N533, N590

Loop3: N582, N608, N611, N617

Loop4: -

Loop5: Q691, Q698

Loop6: N712, N717

Loop7: N764, N775

Loop8: N815, N817, N820

SEQ ID NO 3:

Loop1: -

Loop2: N227, N232

Loop3: N286, N305, N314, N315, N327, N333

Loop4: -

Loop5: Q405

Loop6: -

Loop7: N482, N485, N489, N496, N500, Q513

Loop8: N54, N548, N549, Q553, N555, N560, Q562

SEQ ID NO 4:

Loop1: Q218, Q225

Loop2: Q254, Q257, N258, N261, N266, N270, Q271, N272, N280

Loop3: N322, N348, N358, Q359, N364, N370, N372, N375

Loop4: N408, N412, N421, N424, N428

Loop5: N468, Q471, Q477

Loop6: -

Loop7: N547, N550, N551, Q553, N567, Q572

Loop8: Q615, N617, N618, N619, N622

Modifications in loops 2 and 3 are of particular interest with regard toimproving thermostability. Loop 2 is of interest due to its interactionswith another domain in the N-terminal part of the sequence. Loop 3 is ofinterest due to possible association with a calcium binding site locatedbetween domain A and domain B.

Engineering for Altered Substrate Specificity

When engineering for altered substrate specificity, either or both ofthe first and second parent enzymes may be an isoamylase or apullulanase, although it is of particular interest for purposes of thepresent invention to obtain improved specificity of pullulanases towardshigher molecular weight branched starchy material such as glycogen andamylopectin, in other words a transfer of “isoamylase-like” specificityto a pullulanase, e.g. by means of modifications in the loops 1-8,preferably loops 1, 2, 4 and 5.

For the transfer of isoamylase-like activity to pullulanase, a looptransfer from an isoamylase to a pullulanase is of particular interest,for example by inserting loop 5 from an isoamylase into the site forloop 5 of a pullulanase, or by inserting loop 1 from an isoamylase intothe site for loop 1 of a pullulanase with the numbering indicated inTable 1.

Activity, Sequence Homology and Overall Beta-Strand, Alpha-Helix andLoop Placement in Sequence Knowledge

Activity, either specific activity or specificity, can be transferred topullulanases, using sequence information from e.g. P. amyloderamosaisoamylase (SEQ ID NO: 4) (high isoamylase activity). Also activity,either specific activity or specificity, can be transferred toisoamylases, using sequence information from e.g. B. acidopullulyticuspullulanase (SEQ ID NO: 1). The loops are analysed for specific residuespresent especially in the beginning of the loop sequence, from the endof the beta-strand in isoamylases (or suggested beta-strand inpullulanases).

The suggested changes exemplified below apply to all pullulanases in thehomologous positions corresponding to those of the two pullulanasesdiscussed:

Providing Pullulanase with Isoamylase-Like Activity

This may be provided by substitutions in loop regions following thebeta-strands in B. acidopullulyticus (SEQ ID NO 1) and B. deramificans(SEQ ID NO 2) pullulanase:

After strand: Beta-1 Beta-2 Beta-3 Beta-4 Beta-5 Beta-6 Beta-7 Beta-8 B.acido. D137G H437Y F489G, A M555A G581A I614Y, F N668G E711D Seq. ID.No. 1 T585A, D W672EKQA B. derami. D149G N533Y F585G, A M651A G677AL710Y, F N764G E807D Seq. ID. No. 2 T681A, D W768EKQA

For transfer of the high activity of P. amyloderamosa isoamylase towardshigher molecular weight branched starchy material to R. marinusisoamylase or other isoamylases, or to pullulanases, a sequencealignment is performed as described above. By assessing sequencehomology and taking into consideration the “structure” of the enzymes asdescribed above, strategies for mutation can be deduced.

The transfer of higher activity from P. amyloderamosa is preferablyperformed without losing the thermostability of R. marinus isoamylase inany substantial degree. Although it may generally be difficult to altersubstrate activity without altering thermostability, it is contemplatedthat the present invention will allow the obtainment of a higheractivity while at the same time substantially maintaining the highthermostability in R. marinus isoamylase as well as in the morethermostable pullulanases. This is made possible by aligning isoamylasesand pullulanases to be mutated with the “key alignment” and selectingparent enzymes to be mutated as well as specific amino acid residues andregions to be mutated using information obtained from such alignments ofamino acid sequences.

The list below provides examples of possible mutations, based on theseprinciples, that may be performed to obtain higher activity of R.marinus (SEQ ID NO 3) towards the higher molecular weight starchymaterials.

Mutations for Higher Activity of SEQ ID NO 3

Loop1: K183E, L184Q, H185D, P186T, E187S, V188I, E190A, P191Q Preferred;L184Q, P186T, E187S, P191Q

Loop2: H222Q, A223E, K224T, V225Q, H226N, R228A, H229N, L230D, insertVPN between 231 and 232, E232S, R233D, G234A, L235N, R236Q, N242M,P243T, L244E, C245N, A248S, E250D, P251R Preferred; K224T, V225Q, R228A,P251R

Loop3: G289A, V293T, L294W, insertion of TSSDPTT between 294 and 295,G295A, P296T, T297I, L298Y, F300W, 1303L, R306A, A307T, K310E, A311L,D312T, P313S, N314G, delete P316, R317Q, F318Y, L319F, V320Y, Y322N,T325I, N327A, T328N, L329F, D330N, V331T, G332Y, P334T

Preferred; P296T, R306A, P313S, delete P316, V331T, P334T

Loop4: A404S, A405V, A407G

Loop5: D397A, V398I, P400G, G401N, G402S, V405L, H407G, W410Q, Q411G

Loop6: R418L, Y419F, A422S, V423L, R425Q, F426A, W427Q

Loop7: F469M, E472K, L474V, V475Y

Loop8: L542Y, S543L, Q5446L, H447Q

Site-Directed Mutagenesis

Once an isoamylase or pullulanase encoding DNA sequence has beenisolated, and desirable sites for mutation identified, mutations may beintroduced using synthetic oligonucleotides. These oligonucleotidescontain nucleotide sequences flanking the desired mutation sites. In aspecific method, a single-stranded gap of DNA, the enzyme-encodingsequence, is created in a vector carrying the enzyme gene. Then thesynthetic nucleotide, bearing the desired mutation, is annealed to ahomologous portion of the single-stranded DNA. The remaining gap is thenfilled in with DNA polymerase I (Klenow fragment) and the construct isligated using T4 ligase. A specific example of this method is describedin Morinaga et al., (1984), Biotechnology 2, p. 646-639. U.S. Pat. No.4,760,025 discloses the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into enzyme-encoding DNAsequences is described in Nelson and Long, (1989), AnalyticalBiochemistry 180, p. 147-151. It involves the 3-step generation of a PCRfragment containing the desired mutation introduced by using achemically synthesized DNA strand as one of the primers in the PCRreactions. From the PCR-generated fragment, a DNA fragment carrying themutation may be isolated by cleavage with restriction endonucleases andreinserted into an expression plasmid.

Random Mutagenesis

Random mutagenesis is suitably performed either as localised orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

The random mutagenesis of a DNA sequence encoding a parent enzyme may beconveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present inventionrelates to a method for generating a variant of a parent enzyme, whereinthe variant exhibits improved thermal stability relative to the parent,the method comprising:

(a) subjecting a DNA sequence encoding the parent enzyme to randommutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a hostcell, and

(c) screening for host cells expressing an enzyme variant which has analtered property (e.g. thermal stability) relative to the parent enzyme.

Step (a) of the above method of the invention is preferably performedusing doped primers.

For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) ir-radiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at the positionswhich are to be changed. The doping or spiking may be done so thatcodons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding theglucoamylase enzyme by any published technique, using e.g. PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints. The doping scheme may be made byusing the DOPE program which, inter alia, ensures that introduction ofstop codons is avoided (Jensen, L J, Andersen, K V, Svendsen, A, andKretzschmar, T (1998) Nucleic Acids Research 26:697-702).

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a parent glucoamylase is subjected to PCRunder conditions that increase the misincorporation of nucleotides(Deshler 1992; Leung et al., Technique, Vol. 1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133,1974, pp. 179-191), S. cereviseae or any other microbial organism may beused for the random mutagenesis of the DNA encoding the enzyme by, e.g.,transforming a plasmid containing the parent enzyme into the mutatorstrain, growing the mutator strain with the plasmid and isolating themutated plasmid from the mutator strain. The mutated plasmid may besubsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in agenomic or cDNA library prepared from an organism expressing the parentenzyme. Alternatively, the DNA sequence may be present on a suitablevector such as a plasmid or a bacteriophage, which as such may beincubated with or other-wise exposed to the mutagenizing agent. The DNAto be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to performing the expression step b) or the screening step c).Such amplification may be performed in accordance with methods known inthe art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenizing agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich was carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram positive bacteria such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localized to a part of theparent enzyme in question. This may, e.g., be advantageous when certainregions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Homology to Other Parent Enzyme

In an embodiment, the present invention also relates to variants ofisolated parent polypeptides having an amino acid sequence which has adegree of identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3and SEQ ID NO: 4 of at least about 60%, preferably at least about 70%,preferably at least about 80%, preferably at least about 90%, preferablyat least about 93%, more preferably at least about 95%, even morepreferably at least about 97%, and most preferably at least about 99%,and which have pullulanase or isoamylase activity (hereinafter“homologous polypeptides”). In a preferred embodiment, the homologousparent polypeptides have an amino acid sequence which differs by fiveamino acids, preferably by four amino acids, more preferably by threeamino acids, even more preferably by two amino acids, and mostpreferably by one amino acid from any of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 and SEQ ID NO: 4.

The amino acid sequence homology may be determined as the degree ofidentity between the two sequences indicating a derivation of the firstsequence from the second. “Homology” (identity) may be determined by useof any conventional algorithm, preferably by use of the gap program fromthe GCG package version 8 (August 1994) using default values for gappenalties, i.e., a gap creation penalty of 3.0 and gap extension penaltyof 0.1 (Genetic Computer Group (1991) Programme Manual for the GCGPackage, version 8, 575 Science Drive, Madison, Wis., USA 53711).

Preferably, the parent polypeptides comprise the amino acid sequences ofany of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; orallelic variants thereof; or a fragment thereof that has pullulanase orisoamylase activity.

Fragments of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4are polypeptides having one or more amino acids deleted from the aminoand/or carboxyl terminus of these amino acid sequences.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

In another embodiment, the isolated parent polypeptides havingpullulanase or isoamylase activity are encoded by nucleic acid sequenceswhich hybridize under very low stringency conditions, more preferablylow stringency conditions more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with (i) the nucleic acid sequence of SEQ ID NO: 12 or SEQ IDNO: 13; (ii) a subsequence of (i); or (iii) a complementary strand of(i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.). The subsequence of SEQ ID NO: 12 or SEQ ID NO: 13 may be at least100 nucleotides or preferably at least 200 nucleotides. Moreover, thesubsequence may encode a polypeptide fragment which has pullulanase orisoamylase activity, respectively. The parent polypeptides may also beallelic variants or fragments of the polypeptides that have pullulanaseor isoamylase activity.

The nucleic acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a fragment thereof, maybe used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having pullulanase or isoamylase activity, fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 15, preferably at least25, and more preferably at least 35 nucleotides in length. Longer probescan also be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide having pullulanase or isoamylaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 12 or SEQ ID NO: 13 or subsequences thereof,the carrier material is used in a Southern blot. For purposes of thepresent invention, hybridization indicates that the nucleic acidsequence hybridizes to a nucleic acid probe corresponding to the nucleicacid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13, its complementarystrand, or a subsequence thereof, under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions are detected using X-ray film.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(m) using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standardSouthern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with the sequenceof SEQ ID NO: 12 or SEQ ID NO: 13, or its complementary strand, or asubsequence thereof; and (b) isolating the nucleic acid sequence. Thesubsequence is preferably a sequence of at least 100 nucleotides such asa sequence which encodes a polypeptide fragment which has pullulanase orisoamylase activity.

Contemplated parent polypeptides have at least 20%, preferably at least40%, more preferably at least 60%, even more preferably at least 80%,even more preferably at least 90%, and most preferably at least 100% ofthe pullulanase or isoamylase activity of the mature polypeptide of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

The invention will be further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Donor Organisms

Bacillus acidopullulyticus comprises the pullulanase enzyme encoding DNAsequence of the pulB gene (SEQ ID NO: 13) (Kelly, A. P., Diderichsen,B., Jørgensen, S. And McConnett, D. J. (1994) Molecular genetic analysisof the pullulanase B gene of Bacillus acidopullulyticus. FEMSMicrobiology letters 115, 97-106).

Other Strains

E. coli strain: Cells of E. coli SJ2 (Diderichsen, B., Wedsted, U.,Hedegaard, L., Jensen, B. R., Sjøholm, C. (1990) Cloning of aldB, whichencodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillusbrevis. J. Bacteriol., 172, 4315-4321), were prepared for andtransformed by electroporation using a Gene Pulser™ electroporator fromBIO-RAD as described by the supplier.

B. subtilis PL1801. This strain is the B. subtilis DN1885 with disruptedapr and npr genes (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen,B. R., Sjøholm, C. (1990) Cloning of aldB, which encodesalpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J.Bacteriol., 172, 4315-4321). Competent cells were prepared andtransformed as described by Yasbin, R. E., Wilson, G. A. and Young, F.E. (1975) Transformation and transfection in lysogenic strains ofBacillus subtilis: evidence for selective induction of prophage incompetent cells. J. Bacteriol, 121:296-304.

Plasmids

pMOL944. This plasmid is a pUB110 derivative essentially containingelements making the plasmid popagatable in Bacillus subtilis, kanamycinresistance gene and having a strong promoter and signal peptide clonedfrom the amyL gene of B. licheniformis ATCC14580. The signal peptidecontains a SacII site making it convenient to clone the DNA encoding themature part of a protein infusion with the signal peptide. This resultsin the expression of a Pre-protein which is directed towards theexterior of the cell.

The plasmid was constructed by means of ordinary genetic engineering andis briefly described in the following.

Construction of pMOL944

The pUB110 plasmid (McKenzie, T. et al., 1986, Plasmid 15:93-103) wasdigested with the unique restriction enzyme NciI. A PCR fragmentamplified from the amyL promoter encoded on the plasmid pDN1981 (P. L.Jørgensen et al., 1990, Gene, 96, p 37-41) was digested with NciI andinserted in the NciI digested pUB110 to give the plasmid pSJ2624.

The two PCR primers used have the following sequences:

# LWN5494 5′-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC-3′ # LWN54955′-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAA TGAGGCAGCAAGAAGAT-3′

The primer #LWN5494 inserts a NotI site in the plasmid.

The plasmid pSJ2624 was then digested with SacI and NotI and a new PCRfragment amplified on amyL promoter encoded on the pDN1981 was digestedwith SacI and NotI and this DNA fragment was inserted in the SacI-NotIdigested pSJ2624 to give the plasmid pSJ2670.

This cloning replaces the first amyL promoter cloning with the samepromoter but in the opposite direction. The two primers used for PCRamplification have the following sequences:

#LWN5938 5′-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATGAGGCAGCAAGAAGAT-3′ #LWN5939 5′-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC-3′

The plasmid pSJ2670 was digested with the restriction enzymes PstI andBclI and a PCR fragment amplified from a cloned DNA sequence encodingthe alkaline amylase SP722 (Patent #WO9526397-A1) was digested with PstIand BclI and inserted to give the plasmid pMOL944. The two primers usedfor PCR amplification have the following sequence:

#LWN7864 5′-AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3′ #LWN79015′-AACTGCAGCCGCGGCACATCATAATCGGACAAATGGG-3′

The primer #LWN7901 inserts a SacII site in the plasmid.

Subcloning and expression of pullulanase pulB in B. subtilis. The pulBencoding DNA sequence of the invention was PCR amplified using the PCRprimer set consisting of these two oligo nucleotides:

pu1B.upper.SacII 5′-CAT TCT GCA GCC GCG GCA GAT TCT ACC TCG ACA GAAGTC-3′ pu1B.lower.NotI 5′-GTT GAG AAA A GC GGC CGC TTC TTT AAC ACA TGCTAC GG-3′

Restriction sites SacII and NotII are underlined.

The pulB upper SacII primer is situated just after the signal sequenceof the pulB gene and will after cloning in the pMOL944 vector generate asignal fusion to the amyL signal sequence. The pulB lower primer issituated just after the mRNA terminator of the pulB gene.

Genomic DNA Preparation

Strain Bacillus pullulyticus (ID noxxxx) was propagated in liquid TYmedium. After 16 hours incubation at 30° C. and 300 rpm, the cells wereharvested, and genomic DNA isolated by the method described by Pitcheret al. (Pitcher, D. G., Saunders, N. A., Owen, R. J. (1989). Rapidextraction of bacterial genomic DNA with guanidium thiocyanate. Lett.Appl. Microbiol., 8, 151-156).

Chromosomal DNA isolated from B. pullulyticus as described above wasused as template in a PCR reaction using Amplitaq DNA Polymerase (PerkinElmer) according to manufacturers instructions. The PCR reaction was setup in PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01%(w/v) gelatin) containing 200 μM of each dNTP, 2.5 units of AmpliTaqpolymerase (Perkin-Elmer, Cetus, USA) and 100 μmol of each primer

The PCR reactions was performed using a DNA thermal cycler (Landgraf,Germany). One incubation at 94° C. for 1 min followed by thirty cyclesof PCR performed using a cycle profile of denaturation at 96° C. for 10sec, annealing at 60° C. for 30 sec, and extension at 72° C. for 150sec. Five-μl aliquots of the amplification product was analysed byelectrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance of aDNA fragment size 2.5 kb indicated proper amplification of the genesegment.

Subcloning of PCR Fragment

Fortyfive-μl aliquots of the PCR products generated as described abovewere purified using QIAquick PCR purification kit (Qiagen, USA)according to the manufacturer's instructions. The purified DNA waseluted in 50 μl of 10 mM Tris-HCl, pH 8.5. 5 μg of pMOL944 andtwentyfive-μl of the purified PCR fragment was digested with SacII andNotI, electrophoresed in 0.8% low gelling temperature agarose (SeaPlaqueGTG, FMC) gels, the relevant fragments were excised from the gels, andpurified using QIAquick Gel extraction Kit (Qiagen, USA) according tothe manufacturer's instructions. The isolated PCR DNA fragment was thenligated to the SacII-NotI digested and purified pMOL944. The ligationwas performed overnight at 16° C. using 0.5 μg of each DNA fragment, 1 Uof T4 DNA ligase and T4 ligase buffer (Boehringer Mannheim, Germany).

The ligation mixture was used to transform competent B. subtilis PL2306.The transformed cells were plated onto LBPG-10 μg/ml of Kanamycin −0.1%AZCL-Pullulan-agar plates. After 18 hours incubation at 37° C. cellspositively expressing the cloned Pullulanase were seen as coloniessurrounded by blue halos. One such positive clone was restreaked severaltimes on agar plates as used above, this clone was called PULxxx. Theclone PULxxx was grown overnight in TY-10 μg/ml Kanamycin at 37° C., andnext day 1 ml of cells were used to isolate plasmid from the cells usingthe Qiaprep Spin Plasmid miniprep Kit 427106 according to themanufacturers recommendations for B. subtilis plasmid preparations.

Expression of Pullulanase

PULxxx was grown in 25×200 ml BPX media with 10 μg/ml of Kanamycin in500 ml two baffled shakeflasks for 5 days at 30° C. at 300 rpm.

pu1B seq: (SEQ ID NO: 13)AAAAAATGCTTAATAGAAGGAGTGTAATCTGTGTCCCTAATACGTTCTAGGTATAATCATTTTGTCATTCTTTTTACTGTCGCCATAATGTTTCTAACAGTTTGTTTCCCCGCTTATAAAGCTTTAGCAGATTCTACCTCGACAGAAGTCATTGTGCATTATCATCGTTTTGATTCTAACTATGCAAATTGGGATCTATGGATGTGGCCATATCAACCAGTTAATGGTAATGGAGCAGCATACGAGTTTTCTGGAAAGGATGATTTTGGCGTTAAAGCAGATGTTCAAGTGCCTGGGGATGATACACAGGTAGGTCTGATTGTCCGTACAAATGATTGGAGCCAAAAAAATACATCAGACGATCTCCATATTGATCTGACAAAGGGGCATGAAATATGGATTGTTCAGGGGGATCCCAATATTTATTACAATCTGAGTGATGCGCAGGCTGCAGCGACTCCAAAGGTTTCGAATGCGTATTTGGATAATGAAAAAACAGTATTGGCAAAGCTAACTAATCCAATGACATTATCAGATGGATCAAGCGGCTTTACGGTTACAGATAAAACAACAGGGGAACAAATTCCAGTTACCGCTGCAACAAATGCGAACTCAGCCTCCTCGTCTGAGCAGACAGACTTGGTTCAATTGACGTTAGCCAGTGCACCGGATGTTTCCCATACAATACAAGTAGGAGCAGCCGGTTATGAAGCAGTCAATCTCATACCACGAAATGTATTAAATTTGCCTCGTTATTATTACAGCGGAAATGATTTAGGTAACGTTTATTCAAATAAGGCAACGGCCTTCCGTGTATGGGCTCCAACTGCTTCGGATGTCCAATTACTTTTATACAATAGTGAAACAGGACCTGTAACCAAACAGCTTGAAATGCAAAAGAGTGATAACGGTACATGGAAACTGAAGGTCCCTGGTAATCTGAAAAATTGGTATTATCTCTATCAGGTAACGGTGAATGGGAAGACACAAACAGCCGTTGACCCTTATGTGCGTGCTATTTCAGTCAATGCAACACGTGGTATGATAGTCGATTTAGAAGATACGAATCCTCCTGGATGGAAAGAAGATCATCAACAGACACCTGCGAACCCAGTGGATGAAGTAATCTACGAAGTGCATGTGCGTGATTTTTCGATTGATGCTAATTCAGGCATGAAAAATAAAGGGAAATATCTTGCCTTTACAGAACATGGCACAAAAGGCCCTGATAACGTGAAAACGGGTATTGATAGTTTGAAGGAATTAGGAATCAATGCTGTTCAATTACAGCCGATTGAAGAATTTAACAGCATTGATGAAACCCAACCAAATATGTATAACTGGGGCTATGACCCAAGAAACTACAACGTCCCTGAAGGAGCGTATGCAACTACACCAGAAGGAACGGCTCGCATTACCCAGTTAAAGCAACTGATTCAAAGCATTCATAAAGATCGGATTGCTATCAATATGGATGTGGTCTATAACCATACCTTTAACGTAGGAGTGTCTGATTTTGATAAGATTGTTCCGCAATACTATTATCGGACAGACAGCGCAGGTAATTATACGAACGGCTCAGGTGTAGGTAATGAAATTGCGACCGAGCGTCCGATGGTCCAAAAGTTCGTTCTGGATTCTGTTAAATATTGGGTAAAGGAATACCATATCGACGGCTTCCGTTTCGATCTTATGGCTCTTTTAGGAAAAGACACCATGGCCAAAATATCAAAAGAGCTTCATGCTATTAATCCTGGCATTGTCCTGTATGGAGAACCATGGACTGGCGGTACCTCTGGATTATCAAGCGACCAACTCGTTACGAAAGGTCAGCAAAAGGGCTTGGGAATTGGCGTATTCAACGATAATATTCGGAACGGACTCGATGGTAACGTTTTTGATAAATCGGCACAAGGATTTGCAACAGGAGATCCAAACCAAGTTAATGTCATTAAAAATAGAGTTATGGGAAGTATTTCAGATTTCACTTCGGCACCTAGCGAAACCATTAACTATGTAACAAGCCATGATAATATGACATTGTGGGATAAAATTAGCGCAAGTAATCCGAACGATACACAAGCAGATCGAATTAAGATGGATGAATTGGCTCAAGCTGTGGTATTTACTTCACAAGGGGTACCATTTATGCAAGGTGGAGAAGAAATGCTGCGGACAAAAGGCGGTAATGATAATAGTTACAATGCCGGGGATAGCGTGAATCAGTTCGATTGGTCAAGAAAAGCACAATTTGAAAATGTATTCGACTACTATTCTTGGTTGATTCATCTACGTGATAATCACCCAGCATTCCGTATGACGACAGCGGATCAAATCAAACAAAATCTCACTTTCTTGGATAGCCCAACGAACACTGTAGCATTTGAATTAAAAAATCATGCCAATCATGATAAATGGAAAAACATTATAGTTATGTATAATCCAAATAAAACTGCACAAACTCTCACTCTACCAAGTGGAAATTGGACAATTGTAGGATTAGGCAATCAAGTAGGTGAGAAATCACTAGGCCATGTAAATGGCACGGTTGAGGTGCCAGCTCTTAGTACGATCATTCTTCATCAGGGTACATCTGAAGATGTCATTGATCAAAATTAATATTGATTAAGAAATGATTTGTAAAACATTTAAGTCCATTTACACGGGATACTGTGTAAATGGATTTTAGTTTTATCCGTAGCATGTGTTAAAGAAGTAAATAGTAAATGGCAATTTTarget for the Two Amplifying Primers is IndicatedMedia

TY (as described in Ausubel, F. M. et al. (eds.) “Current protocols inMolecular Biology”. John Wiley and Sons, 1995). LB agar (as described inAusubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”.John Wiley and Sons, 1995).

LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M potassiumphosphate, pH 7.0

AZCL-Pullulan is added to LBPG-agar to 0.5% AZCL-pullulan is fromMegazyme, Australia.

BPX media is described in EP 0 506 780 (WO 91/09129).

Example 2 Purification of Bacillus Acidopullulyticus Pullulanase(Promozyme™)

Bacillus acidopullulyticus pullulanase was purified from a fermentationof B. acidopullulyticus (described in EP 63,909), the pullulanase beingsecreted to the medium.

A filter aid was added to the culture broth, which was filtered througha filtration cloth. This solution was further filtered through a Seitzdepth filter plate, resulting in a clear solution. The filtrate wasconcentrated by ultrafiltration on 10 kDa cut-off polyethersulfonemembranes followed by dialfiltration with distilled water to reduce theconductivity. The pH of the concentrated enzyme was adjusted to pH 4.5.The conductivity of the concentrated enzyme was 0.7 mS/cm.

The concentrated pullulanase was applied to an S-Sepharose FF columnequilibrated in 20 mM CH₃COOH/NaOH, pH 4.5, and the enzyme was elutedwith a linear NaCl gradient (0→0.5M). The pullulanase activity eluted asa single peak. The pooled fractions with pullulanase activity weretransferred to 20 mM KH₂PO₄/NaOH, pH 7.0 on a Sephadex G25 column. Theenzyme was further purified by application to a Q-Sepharose FF columnequilibrated in 20 mM KH₂PO₄/NaOH, pH 7.0. After washing the column, thepullulanase was eluted with a linear NaCl gradient (0→0.5M). Fractionswith pullulanase activity were pooled and the buffer was exchanged for20 mM CH₃COOH/NaOH, pH 4.5, on a Sephadex G25 column. The pullulanasewas then applied to a SOURCE 30S column equilibrated in 20 mMCH₃COOH/NaOH, pH 4.5. After washing the column, the pullulanase activitywas eluted with an increasing linear NaCl gradient (0→0.2M). Fractionswith pullulanase activity were pooled and concentrated on anultrafiltration cell with a 10 kDa cut-off regenerated cellulosemembrane. The concentrated enzyme was applied to a Superdex200 sizeexclusion column equilibrated in 20 mM CH₃COOH/NaOH, 200 mM NaCl, pH4.5. Fractions eluted from the Superdex200 column were analyzed bySDS-PAGE and pure pullulanase fractions were pooled.

The pullulanase migrates on SDS-PAGE as a band with M_(r)=100 kDa.

Other pullulanases and isoamylases may be purified essentially in thesame manner.

Sepharose, Sephadex, SOURCE and Superdex are trademarks owned byAmersham Pharmacia Biotech.

Example 3 Thermostability of Pullulanases and Isoamylases

The thermostability of pullulanases and isoamylses may be tested bymeans of DSC (Differential Scanning Calorimetry). The thermaldenaturation temperature, Td, is taken as the top of the denaturationpeak in thermograms (Cp vs. T) obtained after heating enzyme solutionsat a constant, programmed heating rate.

Experimental

A suitable DSC apparatus, e.g. a DSC II apparatus from Hart Scientific(Utah, USA) may used for the experiments. 50 mM buffered solutions areused as solvent for the enzyme (approx. 2 mg/ml) at either pH 10 (50 mMglycine buffer), pH 7 (50 mM HEPES buffer+10 mM EDTA) or pH 4 (50 mMcitrate buffer). The enzyme may be purified as described above. 750 μlenzyme solution is transferred into standard 1 ml sealable hastelloyampoules (Hart Scientific). Ampoules are loaded into the calorimeter andcooled to 5° C. for 15 min. Thermal equilibration is carried out priorto the DSC scan. The DSC scan is performed at from 5° C. to 95° C. at ascan rate of approx. 90 K/hr. Denaturation temperatures are determinedwith an accuracy of approx. +/−2° C. The results are expressed as top todenaturation peak as a function of pH.

Example 4 Activity

Debranching Activity Assay

The results below show that the specific activity (activity/mg pureenzyme) is highly dependent on the enzyme class. Isoamylases areextremely active towards high molecular weight branched starchy materialsuch as glycogen and amylopectin, whereas pullulanases are very low inactivity towards these substrates. The activity unit reflects the numberof reducing ends which are formed during a 10 min. incubation period.The opposite picture is observed with pullulanases, i.e. low activitytowards high molecular weight branched starchy material such as glycogenand amylopectin but high activity towards e.g. pullulan.

A high activity towards amylopectin and glycogen is particularlypreferable when an enzymatic debranching is to take place together withthe action of an α-amylase in the liquefaction process. On the otherhand, a high activity towards small oligosaccharides such as pullulan ispreferable when an enzymatic debranching is to take place during thesaccharification step, i.e. after the liquefaction process when the highmolecular weight components have been broken down to smalleroligosaccharides. If a pullulanase could be altered to have a highactivity (specificity) towards high molecular weight compounds such asamylopectin, this would be highly preferable when the pullulanase isadded during the liquefaction process.

Substrates used: rabbit liver glycogen and pullulan. Previous tests hadshowed that a high concentration of substrate was needed in order forthe substrate not to be the limiting factor when a linear assay isdeveloped. A “high” substrate concentration is, in this context, 10%w/v. The Somogyi-Nelson assay measures the amount of reducing endsformed by enzymatic degradation of the substrate. With normal assaytimes of up to 3 hours, the formation of reducing ends is fairlylimited, even though the enzyme concentration is high (10% w/v). Thismeans that the assay measures a relatively small difference in reducingends on a very high background which is much higher than the measurabledifference in absorbance during the enzyme treatment. For this reason,the reducing ends in glycogen and pullulan were oxidised with NaBH₄ asfollows in order to reduce the substrate background level:

1000 mg of glycogen was dissolved in 40 ml of water to which 0.2% NaOHhad been added. 800 mg NaBH₄ was added carefully under stirring. Thesolution was stirred for 48 h at 25° C., after which the reaction wasstopped by adding Amberlite IR-118H, a cation exchanger which removesthe boron ions and stop the reaction. The solution was filtered toremove the matrix and was evaporated to give 10 ml. The solution wasdialyased extensively against deionized water in order to removeresidual boron ions. This method was found to reduce the backgroundvalue by at least a factor of 10.

The assay was conducted according to the method of Somogyi-Nelson, using50 mM sodium acetate, pH values of 4.5, 5.0 and 5.5 and a temperature of50° C. (isoamylase) or 60° C. (pullulanases), with a reaction time of 10min. Glucose was used as a standard, a standard curve being made fromsolutions containing of 0-200 mg glucose/liter.

TABLE 3 Glycogen from Rabbit liver Temp. pH PUN/mg Pullulanase from 60°C. 4.5 50 B. acidopullulyticus 60° C. 5.0 49 (SEQ ID NO 1) 60° C. 5.5 51Pullulanase from 60° C. 4.5 37 B. deramificans 60° C. 5.0 31 (SEQ ID NO2) 60° C. 5.5 30 Isoamylase from 50° C. 4.5 2829 Pseudomonas 50° C. 5.02858 (SEQ ID NO 4) 50° C. 5.5 2709 Pullulan Pullulanase from 60° C. 4.5402 B. acidopullulyticus 60° C. 5.0 414 (SEQ ID NO 1) 60° C. 5.5 393Pullulanase from 60° C. 4.5 288 B. deramificans 60° C. 5.0 276 (SEQ IDNO 2) 60° C. 5.5 255 Isoamylase from 50° C. 4.5 14 Pseudomonas 50° C.5.0 14 (SEQ ID NO 4) 50° C. 5.5 6

SEQ ID NO 12: (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 2181 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (vi) ORIGINAL SOURCE: (B) STRAIN: Rhodothermus marinus DSM4252 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1 . . . 2181 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATG TCA CAT AGC GCG CAA CCG GTT ACGTCG GTA CAG GCC GTC TGG CCC 48 Met Ser His Ser Ala Gln Pro Val Thr SerVal Gln Ala Val Trp Pro  1               5                  10                  15 GGC CGG CCTTAT CCG CTG GGT GCC ACC TGG GAC GGG CTG GGC GTC AAC 96 Gly Arg Pro TyrPro Leu Gly Ala Thr Trp Asp Gly Leu Gly Val Asn             20                  25                  30 TTT GCC CTC TACAGC CAG CAC GCC GAG GCG GTC GAA CTG GTG CTG TTC 144 Phe Ala Leu Tyr SerGln His Ala Glu Ala Val Glu Leu Val Leu Phe         35                  40                  45 GAC CAC CCG GAC GATCCC GCG CCT TCG CGC ACG ATC GAA GTG ACC GAA 192 Asp His Pro Asp Asp ProAla Pro Ser Arg Thr Ile Glu Val Thr Glu     50                  55                  60 CGG ACA GGC CCG ATC TGGCAT GTG TAC CTG CCC GGC CTG CGT CCC GGC 240 Arg Thr Gly Pro Ile Trp HisVal Tyr Leu Pro Gly Leu Arg Pro Gly 65                  70                  75                  80 CAG CTCTAC GGC TAT CGC GTC TAC GGA CCC TAC CGG CCG GAG GAA GGC 288 Gln Leu TyrGly Tyr Arg Val Tyr Gly Pro Tyr Arg Pro Glu Glu Gly                 85                  90                  95 CAC CGC TTCAAT CCG AAC AAG GTG CTG CTC GAC CCC TAC GCG AAG GCC 336 His Arg Phe AsnPro Asn Lys Val Leu Leu Asp Pro Tyr Ala Lys Ala            100                 105                 110 ATC GGC CGG CCCCTT CGC TGG CAC GAC AGC CTC TTC GGT TAC AAA ATC 384 Ile Gly Arg Pro LeuArg Trp His Asp Ser Leu Phe Gly Tyr Lys Ile        115                 120                 125 GGC GAT CCG GCC GGGGAT CTG TCG TTC TCC GAA GAA GAC AGC GCT CCG 432 Gly Asp Pro Ala Gly AspLeu Ser Phe Ser Glu Glu Asp Ser Ala Pro    130                 135                 140 TAC GCG CCG CTG GGA GCCGTC GTG GAG GGC TGT TTC GAG TGG GGC GAC 480 Tyr Ala Pro Leu Gly Ala ValVal Glu Gly Cys Phe Glu Trp Gly Asp145                 150                 155                 160 GAC CGCCCG CCG CGC ATT CCC TGG GAA GAC ACG ATC ATC TAC GAA ACG 528 Asp Arg ProPro Arg Ile Pro Trp Glu Asp Thr Ile Ile Tyr Glu Thr                165                 170                 175 CAC GTC AAGGGC ATC ACG AAG CTG CAT CCG GAA GTG CCG GAG CCG CTG 576 His Val Lys GlyIle Thr Lys Leu His Pro Glu Val Pro Glu Pro Leu            180                 185                 190 CGG GGG ACG TATCTG GGG CTG ACC TGC GAG CCG GTG CTG GAG CAC CTG 624 Arg Gly Thr Tyr LeuGly Leu Thr Cys Glu Pro Val Leu Glu His Leu        195                 200                 205 AAG CAG CTG GGC GTCACC ACG ATC CAG CTC CTT CCG GTG CAC GCA AAA 672 Lys Gln Leu Gly Val ThrThr Ile Gln Leu Leu Pro Val His Ala Lys    210                 215                 220 GTG CAC GAT CGG CAC CTGGTC GAG CGC GGC CTG CGC AAC TAC TGG GGC 720 Val His Asp Arg His Leu ValGlu Arg Gly Leu Arg Asn Tyr Trp Gly225                 230                 235                 240 TAC AATCCG CTC TGC TAC TTT GCG CCG GAG CCC GAG TAC GCC ACG AAC 768 Tyr Asn ProLeu Cys Tyr Phe Ala Pro Glu Pro Glu Tyr Ala Thr Asn                245                 250                 255 GGG CCG ATCTCG GCC GTG CGC GAG TTC AAG ATG ATG GTG CGG GCG CTG 816 Gly Pro Ile SerAla Val Arg Glu Phe Lys Met Met Val Arg Ala Leu            260                 265                 270 CAT GCT GCC GGCTTC GAG GTG ATC GTC GAC GTG GTC TAC AAC CAC ACG 864 His Ala Ala Gly PheGlu Val Ile Val Asp Val Val Tyr Asn His Thr        275                 280                 285 GGC GAA GGC GGC GTGCTG GGC CCC ACG CTG TCG TTC CGG GGC ATC GAC 912 Gly Glu Gly Gly Val LeuGly Pro Thr Leu Ser Phe Arg Gly Ile Asp    290                 295                 300 AAC CGC GCC TAC TAC AAGGCC GAT CCG AAC AAC CCG CGC TTT CTG GTC 960 Asn Arg Ala Tyr Tyr Lys AlaAsp Pro Asn Asn Pro Arg Phe Leu Val305                 310                 315                 320 GAT TACACG GGC ACC GGC AAC ACG CTG GAC GTG GGC AAC CCC TAC GTC 1008 Asp Tyr ThrGly Thr Gly Asn Thr Leu Asp Val Gly Asn Pro Tyr Val                325                 330                 335 ATC CAG CTCATC ATG GAC AGC CTG CGC TAC TGG GTC ACT GAA ATG CAC 1056 Ile Gln Leu IleMet Asp Ser Leu Arg Tyr Trp Val Thr Glu Met His            340                 345                 350 GTC GAC GGC TTTCGG TTC GAC CTG GCC GCC GCG CTG GCC CGC GAG CTG 1104 Val Asp Gly Phe ArgPhe Asp Leu Ala Ala Ala Leu Ala Arg Glu Leu        355                 360                 365 TAC GAC GTG GAC ATGCTC TCG ACC TTT TTT CAG GTC ATT CAG CAG GAC 1152 Tyr Asp Val Asp Met LeuSer Thr Phe Phe Gln Val Ile Gln Gln Asp    370                 375                 380 CCG GTG CTC AGC CAG GTCAAG CTC ATC GCC GAA CCC TGG GAC GTC GGG 1200 Pro Val Leu Ser Gln Val LysLeu Ile Ala Glu Pro Trp Asp Val Gly385                 390                 395                 400 CCG GGGGGG TAT CAG GTG GGA CAT TTT CCC TGG CAG TGG ACC GAG TGG 1248 Pro Gly GlyTyr Gln Val Gly His Phe Pro Trp Gln Trp Thr Glu Trp                405                 410                 415 AAC GGC CGCTAT CGT GAC GCC GTG CGC CGC TTC TGG CGG GGC GAT CGG 1296 Asn Gly Arg TyrArg Asp Ala Val Arg Arg Phe Trp Arg Gly Asp Arg            420                 425                 430 GGC CTC AAC GGTGAG TTT GCC ACG CGC TTT GCC GGC TCC AGC GAT CTG 1344 Gly Leu Asn Gly GluPhe Ala Thr Arg Phe Ala Gly Ser Ser Asp Leu        435                 440                 445 TAC GAA CGT AGC GGTCGT CGT CCG TTC GCT TCG ATC AAC TTC GTC ACG 1392 Tyr Glu Arg Ser Gly ArgArg Pro Phe Ala Ser Ile Asn Phe Val Thr    450                 455                 460 GCG CAC GAC GGC TTC ACGCTG GAA GAC CTG GTC AGC TAC ACG AAA AAG 1440 Ala His Asp Gly Phe Thr LeuGlu Asp Leu Val Ser Tyr Thr Lys Lys465                 470                 475                 480 CAC AACGAA GCG AAT CTG GAA GGC AAC CGG GAC GGC ATG GAC GAA AAC 1488 His Asn GluAla Asn Leu Glu Gly Asn Arg Asp Gly Met Asp Glu Asn                485                 490                 495 TAC AGC ACGAAC TGC GGG GTG GAG GGA CCC ACG CAG GAT CCG TCC GTG 1536 Tyr Ser Thr AsnCys Gly Val Glu Gly Pro Thr Gln Asp Pro Ser Val            500                 505                 510 CTG GCC TGC CGGGAA GCG CTC AAG CGC AGC CTG ATC AGC ACG CTC TTT 1584 Leu Ala Cys Arg GluAla Leu Lys Arg Ser Leu Ile Ser Thr Leu Phe        515                 520                 525 CTC TCG CAG GGC GTGCCC ATG CTG CTG GGC GGC GAC GAG CTG TCG CGC 1632 Len Ser Gln Gly Val ProMet Leu Leu Gly Gly Asp Glu Leu Ser Arg    530                 535                 540 ACG CAG CAC GGC AAC AACAAC GCC TAT TGC CAG GAC AAC GAG ATC AGC 1680 Thr Gln His Gly Asn Asn AsnAla Tyr Cys Gln Asp Asn Glu Ile Ser545                 550                 555                 560 TGG TACAAC TGG CAG CTC GAC ACG CGC AAG CAG CAG TTT CTG GAG TTC 1728 Trp Tyr AsnTrp Gln Leu Asp Thr Arg Lys Gln Gln Phe Leu Glu Phe                565                 570                 575 GTG CGC CAGACG ATC TGG TTT CGC AAG CAG CAT CGG AGC TTC CGG CGC 1776 Val Arg Gln ThrIle Trp Phe Arg Lys Gln His Arg Ser Phe Arg Arg            580                 585                 590 CGC CAT TTT CTGACC GGA TTG CCC AAC GGC GGA AGG CCC CGA CGC AGT 1824 Arg His Phe Leu ThrGly Leu Pro Asn Gly Gly Arg Pro Arg Arg Ser        595                 600                 605 CTG GTG GCA CCT GAGGGT CGG CCC ATG CGC CAC GAG GAC TGG ACC AAC 1872 Leu Val Ala Pro Glu GlyArg Pro Met Arg His Glu Asp Trp Thr Asn    610                 615                 620 CCG GAG CTG ACG GCC TTCGGA CTG CTG CTG CAC GGC GAC GCC ATT CAG 1920 Pro Glu Leu Thr Ala Phe GlyLeu Leu Leu His Gly Asp Ala Ile Gln625                 630                 635                 640 GGG ACCGAC GAG CAC GGA CGA CCG TTT CGC GAC GAC ACG TTT CTG ATT 1968 Gly Thr AspGlu His Gly Arg Pro Phe Arg Asp Asp Thr Phe Leu Ile                645                 650                 655 CTG TTC AACAAC GGC AGC GAA GCC GTG CCG GTC GTG GTG CCG GAG GTA 2016 Leu Phe Asn AsnGly Ser Glu Ala Val Pro Val Val Val Pro Glu Val            660                 665                 670 TGC TCC TGT GGCAAG CCG CAC CAC TGG GAG GTG GTC CCG GTG TTT CAA 2064 Cys Ser Cys Gly LysPro His His Trp Glu Val Val Pro Val Phe Gln        675                 680                 685 CGC AAT GTG GAG CCCCCC ACG TGC GCG CCC GGC GAG ACG CTG TCG CTC 2112 Arg Asn Val Glu Pro ProThr Cys Ala Pro Gly Glu Thr Leu Ser Leu    690                 695                 700 CCG CCC GGC GTG CTG ACGGTG CTG GTG GCC GTA CCG CCG TTC TCG GAT 2160 Pro Pro Gly Val Leu Thr ValLeu Val Ala Val Pro Pro Phe Ser Asp705                 710                 715                 720 GGA AACACG GAG CCG GCC TGA 2181 Gly Asn Thr Glu Pro Ala *                 725

1. An isolated variant of a parent pullulanase, wherein the variant (a)has a degree of sequence identity to SEQ ID NO: 2 of at least 95% whensequence identity is determined using GAP (version 8), using a gapcreation penalty of 3.0 and a gap extension penalty of 0.1, (b)comprises a substitution at one or more positions corresponding topositions 515-554 of SEQ ID NO: 2, and (c) has pullulanase activity. 2.The variant of claim 1, which comprises a substitution at a positioncorresponding to position 522 of SEQ ID NO:
 2. 3. The variant of claim1, which comprises a substitution at a position corresponding toposition 533 of SEQ ID NO:
 2. 4. The variant of claim 1, furthercomprising a substitution at one or more positions corresponding topositions 465-493, 515-554, 580-621, 649-664, 680-711, 714-717, 757-765and 804-834 of SEQ ID NO:
 2. 5. The variant of claim 1, which has adegree of sequence identity to SEQ ID NO: 2 of at least 97%.
 6. Thevariant of claim 1, which has a degree of sequence identity to SEQ IDNO: 2 of at least 99%.
 7. The variant of claim 1, wherein the parentpullulanase is SEQ ID NO:
 2. 8. A method for converting starch to one ormore sugars, comprising (a) liquefying the starch with at least onevariant of claim 1 and at least one alpha-amylase to form dextrin; and(b) saccharifying the dextrin with at least one glucoamylase to form theone or more sugars.